Coronaviruses cause a variety of important acute and chronic diseases of man and animals ranging from mild respiratory infections through fatal neonatal diarrhea, hepatitis, wasting disease, and neurologic diseases. In recent years significant advances in the understanding of the molecular biology of the coronaviruses have been made using the A59 strain of MHV as a model system. The strategy of coronavirus transcription is unique compared to the transcription strategy used by other plus stranded viruses. One of the interesting features of coronaviruses RNA structure and transcription is the evidence that a small leader RNA is added to the 5' ends of the viral mRNAs. Important new advances in this interesting system can be made by applying the techniques I have used to study the small leader RNA of vesicular stomatitis virus (VSV) and its possible host shut-off functions to the study of the coronavirus small RNA and its role in coronavirus replication. In the second area of study I will use a cell-free system used to study VSV replication for the study of coronavirus transcription. The specific areas of study for this protocol include: 1) a synthetic oligonucleotide complementary to the 5' end of viral mRNA 6 and 7 will be used to clone the leader region of coronaviruses into a single-stranded bacteriophage, 2) the clones will be used to determine the location and frequency of the leader sequence on the genomic and subgenomic RNAs, 3) the clones will be used as sensitive probes for the detection and characterization of both the plus and minus sense coronavirus leader RNAs, 4) the small RNA will be examined to determine whether it is associated with any cellular or viral proteins, 5) the kinetics of appearance of the small RNA will be determined, 6) The ribosome binding sites and the degree of methylation at the 5 feet end of the viral genomic and certain subgenomic mRNAs will be determined, 7) an assay used to study VSV replication will be optimized for the study of coronaviurs transcription, 8) antibodies to purified coronavirus structural and non-structural proteins will be added to determine if the leader RNA is made in vitro and to study the mechanisms of coronavirus transcription.